The present invention relates generally to lymphotoxins and lymphoblastoid cell lines useful in production of lymphotoxins. More particularly, the present invention relates to the discovery of two new lymphotoxins which have antitumor activity. The present invention also relates to the production of useful quantities of these two lymphotoxins by stimulation of the HUT-102 continuous human cell line.
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
It is well known that lymphocytes can be stimulated by co-culture with antigens or mitogens in vitro to release a complex family of soluble effector molecules known as lymphokines(1). Lymphotoxins (LT) are a type of lymphokine which are cytotoxic for cells in vitro. Various LT's have been shown to selectively attack neoplastic cells in preference to primary cells in vitro. As a result, much interest has been generated in isolating and studying LT molecules due to their possible use as antitumor agents.
In the past, studies of LT have been difficult because LT forms found in lymphocyte supernatant have been shown to be functionally and physically heterogeneous (2, 3). The past studies have been conducted with unseparated populations of lymphocytes which leads to the heterogeneity of the LT forms found in the supernatant. The various LT forms or molecular weight classes which have been found in lymphocyte supernatant include complex (CX), alpha (.alpha.), beta (.beta.) and gamma (.gamma.). The LT classes are identified and classified by molecular weight (mw) as follows: CX=200,000 daltons (d) or greater; alpha=70,000-90,000 d; beta=30,000-50,000 d and gamma=15,000-25,000 d (2).
The problem regarding heterogeneous LT forms in lymphocyte supernatants has been partially resolved by the finding that different classes of human lymphocytes can be stimulated in vitro to release different and common LT forms (1, 4). Human natural killer cells (NK) stimulated with lectins or contacted with sensitive target cells, release LT forms termed NK-LT that are functionally different from other LT forms for they appear to be species specific, bind and lyse NK sensitive target cells (K-562 and MOLT-4), but do not lyse NK resistant cells (Raji) in vitro (5, 6). Freshly isolated human T cells can be stimulated with lectin to release large MW LT forms such as precursor alpha heavy (140,000-160,000 d) and alpha heavy (140,000-120,000 d) that are unique to T cells. Upon mild denaturation, these forms can be reduced into the smaller molecular weight LT classes (4). Also, continuous human B cell lines appear to release a very restricted MW class of alpha LT with a MW of 90,000-100,000 d.
There is presently a continuing need to isolate and identify new LT's which are useful as antitumor agents and to develop methods for producing supernatants having relatively large, homogeneous amounts of the LT for isolation and use as an antitumor agent.